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Size-exclusion-based extraction of affinity ligands and active compounds from natural samples

a technology applied in the field of size exclusion-based extraction of affinity ligands and active compounds from natural samples, can solve the problems of difficult to isolate any single active compound, difficult to isolate active compound, difficult to isolate active compound, etc., and achieve the effect of rapid isolation and characterization

Inactive Publication Date: 2007-02-20
BBM HLDG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

"The present invention is a method that combines affinity interactions with size exclusion methods to quickly isolate and characterize small molecule compounds from complex mixtures such as natural samples. The method allows for simultaneous screening of multiple compounds and can be used with various analytical techniques for further analysis and characterization. The method involves mixing a protein target with a natural sample, removing unbound small molecule compounds, dissociating the complex, and isolating the free ligand from the protein target and other large molecules. The isolated ligand can then be subjected to analytical techniques for identification. The technical effects of the invention include improved efficiency and accuracy in the isolation and characterization of small molecule compounds from complex biological materials."

Problems solved by technology

Once an active sample is identified, however, it can be difficult to isolate the active compound, particularly from natural product extracts or natural samples.
Natural samples such as natural extracts represent a highly and chemically diverse collection of compounds that include very small to very large molecules, which makes it very difficult to isolate any single active compound.
However, these methods have their limitations, so that a need remains, too, for a successful analytical methodology that provides high throughput screening of combinatorial libraries against biological targets for identification of active ligands.
On the other hand, limitations associated with heterogeneous assays create a demand for a breakthrough technology for rapid and efficient screening of natural samples screened in solution.
Solution-phase assays are desirable to increase screening specificity, but current methodologies involve iterative processes that are long and laborious.
These attempts to screen combinatorial libraries and other complex mixtures of compounds on the basis of size-exclusion separation have had limited success in actual application, particularly under the rigors of high throughput screening.
One major problem has been the tendency of transfer lines and other conduits used in high-throughput, size-exclusion-complex isolation screening protocols, particularly transfer lines to reverse phase HPLC column, to become clogged and impassable after only 1-2 hours of operation.
Such clogging, which occurs due to irreversible collection of the protein on the stationary phase of the HPLC column, necessitates frequent changes of the transfer lines.
It also is a limiting factor in the overall effectiveness and on-line automation of such screening protocols.

Method used

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  • Size-exclusion-based extraction of affinity ligands and active compounds from natural samples
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  • Size-exclusion-based extraction of affinity ligands and active compounds from natural samples

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embodiment

Competitive Binding Embodiment

[0041]Another embodiment of the invention combines the use of competitive binding along with the described screening assay. Utilization of a known competitive ligand (CL) that binds to a selected target allows determination of whether the active ligand extracted from a biological mixture is bound to a specific binding site or known site of the target protein. In this case, a CL that binds to a known site is added to the reaction mixture of target and biological sample containing a small molecular weight affinity ligand at the first, mixing and incubation stage. The resulting mixture of target / natural sample / known competitive ligand can be subjected to the two size-exclusion steps of the invention, as previously described. As needed, a dissociation step may be performed as previously discussed, to remove protein prior to analysis of any detected small molecule ligands. During mass spectrometry, MS-LC, or whatever analysis is performed, both ligand (L) an...

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Off-line Format)

[0050]The off-line screening procedure of the invention is demonstrated using human carbonic anhydrase II (CAII) as a target, and a natural sample (NS) inactive toward CAII (e.g., inert fungal extract) that is spiked with a known, small molecule ligand that binds specifically to the active site of CAII, acetazolamide (AZ). Approximately 20 μM (micromolar) of CAII was mixed and incubated with approximately 1 microgram of an inactive natural sample containing about 10 μM (micromolar) of AZ in a buffered solution such as phosphate buffered saline, pH 7.0, 1% DMSO, for final CAII and AZ concentrations of about 1 micromolar. Size-exclusion chromatography was performed using a Pharmacia HR 10 / 10 column at 4 ml / min using a mobile phase of 200 mM ammonium acetate, pH 7.0. Typically, the excluded volume containing the target protein (CAII) with bound ligand (AZ), elutes from the column in about 0.6-0.7 minutes under such conditions.

[0051]FIG. 3A shows an LC-MS chromatogram of...

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Abstract

The invention encompasses an improved, rapid, size-exclusive method for screening for small molecular weight ligands that bind specifically to a protein target, using size-exclusion separation, ultrafiltration, and mass spectrometry.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application claims benefit of U.S. Provisional Application 60 / 119,966, filed Feb. 12, 1999, and International Application No. PCT / US00 / 03562, filed Feb. 11, 2000, the whole of which are hereby incorporated by reference herein.BACKGROUND OF THE INVENTION[0002]The present invention relates to screening complex biological materials, such as natural samples, natural products, and combinatorial libraries, for active compounds such as affinity ligands. In particular, active compounds are extracted using size-exclusion separation and ultrafiltration, and are subjected to subsequent analysis. The analysis may include structural or functional characterization of an isolated active compound by any one of many techniques known to one of ordinary skill in the art of screening complex biological materials. Suitable analytical techniques include, but are not limited to, mass spectrometry, liquid and other chromatography, a secondary assay, and the...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C12Q1/68B01D15/08G01N27/62G01N30/02G01N30/14G01N30/46G01N30/72G01N30/88G01N33/15G01N33/50G01N33/538G01N33/566G01N33/68
CPCG01N33/538G01N33/68G01N33/6848G01N33/6845B01J2219/00707G01N30/02G01N30/468G01N30/7233B01D15/34B01D15/3804
Inventor DUNAYEVSKIY, YURIY M.HUGHES, DALLAS E.WEISKOPF, ANDREW S.
Owner BBM HLDG